Compounds and methods for generating oxygen and free radicals used in general oxidation and reduction reactions

ABSTRACT

Composition and methods; for carrying out a variety of oxidation reactions, reduction reactions, or both utilizing a free radical generating catalyst, a mediator, and a reductant are disclosed. The free radical generating catalyst is generally a peroxidase obtained from living organisms, such as white rot fungi. Suitable peroxidases include lignin peroxidase, horse radish peroxidase, and lactoperoxidase. Suitable mediators include veratryl alcohol, iodine, methoxybenzenes, Mn II, and ABTS. Suitable reductants include EDTA, oxalate, hydroquinones, quinones plus quinone reductase, and hydrogen peroxide.

This work was supported by a grant from the National Institute ofHealth, Grant No. ESO4922.

This application is a continuation of application Ser. No. 08/001,106,filed Jan. 5, 1993, now U.S. Pat. No. 5,389,356.

BACKGROUND

1. Field of the Invention

This invention describes a method for catalyzing oxidation reactions,reduction reactions, or both using free radical intermediates. Morespecifically, this invention involves the use of biologically derivedperoxidases in the generation of a variety of oxidation or reductionagents consisting of cation radicals, anion radicals, neutral radicals,or oxygen radicals. Such oxidation and reduction agents can be employedseparately or in combination to carry out a wide variety of oxidation orreduction reactions, some of which involve the degradation ofrecalcitrant organic compounds such as organic environmental pollutants.

2. The Relevant Technology

The use of oxidation or .reducing agents to carry out oxidations orreductions on targeted substrates is mature technology well-known in theart. Desired oxidation and reduction reactions can be carried out on amultitude of different substrates simply by reacting the substrate witha stoichiometrically adequate amount of an appropriate oxidant orreductant. Commonly used oxidants or reductants which can be produced ina commercially feasible manner include a wide variety of generallyinorganic agents. The feasibility of using such oxidants or reductantsis often limited by such restraints as the cost of the reactant inrelation to the value of the reacted substrate, the ability to controlthe reaction, and the ability to obtain suitable concentrations of thereacted substrate in reasonably pure amounts.

More complicated oxidation and/or reduction reactions have been createdwhich involve organic intermediates, such as hydroquinones,alkylanthraquinones, anilines, hydrazines, or metal complexed chelatingagents. In some cases, the reactant is a catalyst which is continuouslyregenerated. For example, U.S. Pat. No. 5,143,710 to Sawyer et al.discloses methods for generating superoxide ions in situ catalyzed byaniline, N-substituted aniline compounds, or phenylhydrazine compounds.The superoxide ion, which is an anion radical, is useful for a number ofdifferent applications discussed within Sawyer et al. Superoxide ionshave proven particularly effective in destroying a variety ofhalogenated hydrocarbons such as polychlorinated biphenyls ("PCBs") andsimilar toxic materials. In general, superoxide ions are useful reducingagents.

U.S. Pat. No. 3,998,936 to Ernst et al. discloses a process forregenerating the activity of the catalyst used in the hydrogenation (orreduction) stage of the cyclic anthraquinone process for producinghydrogen peroxide involving the use of a platinum group metal catalyst.However, Ernst et al. does not disclose how an overalloxidation/reduction system could be constructed that would have broadapplication.

U.S. Pat. No. 4,751,068 to Bicker et al. discloses a method ofcatalyzing oxidation/reduction reactions of simple molecules through theredox catalytic activity of chelating agents complexed with a metal atom(the complex being referred to as a "chelate"). These chelates have beenshown to be useful in converting CO and H₂ O to CO₂, CO and H₂ S to COS,CS and H₂ S to CS₂, CO and NH₃ to CONH, and CO and RNH₂ to RNCO.However, in order to regenerate the spent chelates it is necessary toreact the chelates with oxidants or reductants. No self-sustainingreaction sequence is disclosed in Bicker et al.

More recently, with the advent of more refined biochemical techniques,biologically induced oxidations and reductions have been carried outusing, e.g., fungi and agents which are secreted thereby. Thesebiologically derived reactions are often superior to simply addingoxidation and/or reducing agents to a reaction mixture because of theirlower cost and greater ability to more carefully control the reactionconditions, especially those reactions which involve the use of enzymes.Enzymes have the advantage being able to overcome high reaction barrierswithout the input and/or generation of large amount amounts of energysuch as heat. In addition, as long as the biological agent is kept aliveby ensuring that the system has adequate quantities of nutrients (someor all of which are supplied by the chemicals targeted for degradation)it will continue to produce adequate quantities of the oxidation orreduction agents. In this manner, the reaction is often self-sustainingso that no new reactants need to be added to complete the oxidationand/or reduction reactions.

There are numerous examples of biologically induced degradation oforganic molecules. For example, lignin, which is the structural polymerfound in wood and a substance which is otherwise highly resistant tomany forms of biodegradation, is readily degraded in the presence of thewhite rot fungus Phanerochaete chrysosporium. Kirk, T. et al., Arch.Microbiol. 117:277-85 (1978). Lignin degradation is catalyzed by a groupof enzymes including extracellular peroxidases secreted by P.chrysosporium under nutrient nitrogen-limiting conditions. Gold, M. etal., Arch. Biochem. Biophys., 234:353-62 (1984); Tien, Met al., Proc.Natl. Acad. Sci. USA, 81:2280-84 (1984). It is known that both ligninperoxidases ("LIP") and manganese-dependent peroxidases are produced bywhite rot fungi. Glenn, J. et al, Arch. Biochem. Biophys., 242:329-41(1985). The fungi also produce enzymes that generate hydrogen peroxide.Kelley, R. et al, Arch. Microbiol., 144:248-53 (1986); Kersten, P.,Biochemistry, 87:2936-40 (1990). Veratryl alcohol (3,4-dimethoxybenzylalcohol) is a secondary metabolite of P. chrysosporium and is alsobelieved to be involved in lignin degradation. Harvey, P. et al., FEBSLett., 195:242-46 (1985).

In addition, the degradation of several environmental pollutants tocarbon dioxide by white rot fungi has also been reported. U.S. Pat. No.4,891,320 to Aust et al; Bumpus, J. et al., Science, 228:1434-36; Ryan,T. et al., Appl. Microbiol. Biotechnol., 31:302:07 (1989); Fernando, T.et al, Appl. Microbiol. Biotechnol., 56:1666-71 (1990); Kennedy, D. etal., Appl. Microbiol. Biotechnol., 56:2346-53 (1990).

Although these articles generally discuss the use of white rot fungi todegrade lignin, no specific information as to the mechanism of thisdegradation is revealed. To the extent that certain intermediatesubstances such as LiP or veratryl alcohol have been shown to beinvolved, these articles do not contain information that would teach howto utilize white rot fungi or other useful organisms in a variety ofoxidation or reduction reactions to generally oxidize or reduce anyorganic compound.

In the last few decades, there has been growing concern about theaccumulation of toxic organic pollutants in the soil and water. Manyindustrial operations, particularly those involving chemical processes,have resulted in the contamination of huge amounts of soil, which inturn pollutes ground water and streams. With the fairly recent passageof stricter environmental legislation mandating the cleanup of what arereferred to as "remediation sites" there has arisen a great need forpractical and economically viable methods of soil and water remediation.

In the case of toxic organic pollutants such as chlorinatedhydrocarbons, PCBs, and other organic solvents, the primary method ofremoving these from the soil involves the temporary removal of thecontaminated soil, which is then passed through large columns throughwhich hot air is passed. This causes the volatile contaminants to bedriven off by evaporation. However, not only is this method extremelyexpensive, it does not guarantee the removal of the pollutants from theenvironment but simply shifts them from the ground into the air. Whilesome degradation of these pollutants may occur in the presence ofsunlight, many of the less reactive compounds are simply scattered intothe air where they might later precipitate back into the earth, albeitin a more diluted form.

While organisms such as white rot fungi have been shown to degradecertain toxic pollutants in the laboratory, their use as general agentsto clean up such toxic pollutants has been limited due to the lack ofunderstanding of the reaction mechanisms involved in their oxidation andreduction. In addition, because many of the most persistent pollutantsexist deep beneath the earth it has not been possible to sustain livingwhite rot fungi or other organism cultures in the highly anaerobicconditions which exist beneath the earth. Finally, without anunderstanding of the necessary intermediates, or "diet" of the reactionsinvolving the degradation of certain organic materials through the useof such organisms it has heretofore been impossible to control, or evenpredict, the types of organic substances that might be degraded throughsuch mechanisms.

From the foregoing it should be understood that what are needed arecompositions and methods which can be generally employed to carry outany number of oxidations or reductions on any targeted organicsubstrate. Moreover, it will be appreciated that it would be asignificant advancement of the art if such compositions and methodscould be cheaply and easily carried out by using relatively inexpensiveraw materials, such as those used to grow white rot fungi.

It would yet be a significant improvement over the prior art if suchcompositions and methods could be varied to alternatively reduce,oxidize, or both, depending on the substrates to be degraded.Specifically, it would be a major advancement in the art if both theoxidative and reductive properties could be carefully controlled so thatcompounds requiring both oxidation and reduction for their degradationcan be fully degraded utilizing a single reactive system, or differentsystems or conditions in series.

It would yet be a significant improvement over the prior art to providecompositions and methods under a variety of conditions which coulddegrade a variety of recalcitrant environmental pollutants such as PCBs,chlorinated hydrocarbons, and other toxic organic wastes without havingto physically alter the reaction conditions once the reactions are setin motion. In addition, because living organisms are typically employedto carry out these reactions, it would be a major advancement in the artif such compositions and methods resulted in the generation ofsufficient molecular oxygen so that the organisms would stay alive evenunder extremely anaerobic conditions, such as in remediation sites wherethe organisms are injected deep into the contaminated soil.

Such compositions and methods are disclosed and claimed herein.

BRIEF SUMMARY AND OBJECTS OF THE INVENTION

The present invention involves the use of free radical generatingcatalysts to initiate a variety of different, yet related, oxidationand/or reduction reactions. Free radicals can be formed by chemical,biological, photochemical or electrochemical methods. A preferred methodof forming the free radicals involves the use of living organism, suchas white rot fungi, which are known to secrete LiP, a free radicalgenerating catalyst. The LiP is usually activated by an electronacceptor such as H₂ O₂ and then reacts with an oxidant precursor (or"mediator) to form the free radical of the mediator. The free radical ofthe mediator is a strong oxidizing agent which can be used to generallycarry out a number of desired oxidation reactions.

For example, the free radical of the mediator can be used to directlyoxidize, and thereby degrade, a targeted substrate. In addition, thefree radical of the mediator can be used to generate molecular oxygen,which is often desired in order to sustain the life of an organism iforganism driven reactions are involved. Finally, the free radical of themediator can be used to form the free radical of certain reductants,which are strong reducing agents. Thus, depending upon the constituentsin the reaction site, any or all of these various reactions can besimultaneously carried out. Moreover, because most of the necessaryreaction constituents are secreted by white rot fungi, such reactionscan be substantially self-sustaining, although it may be important toinitially adjust the concentrations of the required substances for anygiven reaction system.

Preferred free radical generating catalysts include LiP and MnP secretedby white rot fungi, horse radish peroxidase, and lactoperoxidase.Preferred mediators include veratryl alcohol, also secreted by white rotfungi and Mn II. In addition, methoxybenzenes (such as1,2,3-trimethoxybenzene, 1,4-dimethoxybenzene, 1,2,4-trimethoxybenzene),iodide, and ABTS can be used in place of veratryl alcohol.

The reaction of the free radical generating catalyst and the statedmediators yields the veratryl alcohol cation radical, Mn III, the ABTScation radical, or the cation radical of the methoxybenzene involved. Inmost cases, the free radical of the mediator can be used to carry out avariety of oxidation reactions. However, in the case of the iodideradical, iodination of organic molecules can occur unless a suitablereductant, as more fully discussed below, is added to arrest theiodination process.

Preferred reductants include organic acids such as oxalic acid, EDTA,hydroquinones, and the superoxide molecule, which is converted tomolecular oxygen by this scheme. In the case of organic acids orhydroquinones, these reductants are converted into their free radicalanalogs, which can be used to reduce any number of electron acceptors.

In one embodiment of the present invention, the free radical of thereductants are used to reduce chlorinated hydrocarbons such as CCl₄,which results in its dechlorination. However, the present reaction couldbe used to generally dehalogenate any number of halogenated organicsubstances, such as PCBs, DDT, TCDD, etc.. Other organic substanceswhich can be reduced include NBT and cytochrome c.

In addition, the free radical of the reductants can react with molecularoxygen to form the superoxide anion radical. If ferric iron is present,it is reduced to ferrous iron which can generate hydroxyl radical, awell known, powerful oxidant, via the Haber-Weiss reaction.

Therefore, through a series of simultaneous or sequentially inducedreduction and oxidation reactions, molecules such as PCBs can first bedehalogenated in a reduction reaction, and then oxidized to CO₂ in anoxidation reaction. Because the free radical of the mediator is reducedby the reductant, the mediator is generally continuously regeneratedduring a reaction involving such reductants. However, because the anionradical of the organic acid is further oxidized during thedechlorination reaction, which often results in the decarboxylation ofthe organic acid and the generation of CO₂ gas, the organic acid is thusdepleted during this reaction scheme. However, because one of themetabolic byproducts of white rot fungi is oxalic acid, it is oftenpossible to sustain the reaction scheme without having to continuouslyadd additional oxalate ions.

Following the general scheme set forth above it is possible tospecifically produce particular radicals having particular redoxpotentials useful for catalyzing a wide variety of different oxidationor reduction reactions. In particular, the compositions and methods ofthe present invention allow for the production of superoxide, hydroxyl,iodide, organic acid, and semiquinone radicals. Each of these radicalshave different redox potentials suitable for catalyzing any number ofoxidative, reductive, or both types of reactions. As a result, thecompositions and methods of the present invention are capable ofdegrading a wide variety of chemicals (from highly oxidized to highlyreduced).

Specifically, anion radicals of, for example oxalate or EDTA, have beenshown to be useful in the degradation of CCl₄. Superoxide is known toreduce molecules like DDT, while hydroxyl radicals are known to be themost powerful oxidants. PCBs and many other recalcitrant chemicals areknown to be oxidized by hydroxyl radicals.

The methods of the present invention are far more economical thanpreviously known methods for carrying out similar oxidation and/orreduction reactions as they can be carried out at room temperature andatmospheric pressure. Furthermore, the raw materials necessary to carryout the reactions of the present invention are very inexpensive incomparison to previously known methods of oxidizing, for example, toxicpollutants such as CCl₄, PCBs, DDT, and other recalcitrant organicmaterials.

More importantly, because the mechanisms of the various reactionsinvolved in the present invention are understood they can more easily becontrolled. This includes both the rate as well as the particularreaction that is involved, including being able to choose the targetedsubstrate to be oxidized, reduced, or otherwise degraded. Thus, bysimply altering the identities and concentrations of the raw materialsadded to a reaction site, as well as the pH, the process can be tailoredto degrade a wide variety of organic substances, including halogenatedhydrocarbons.

Moreover, none of the raw materials used in the methods of the presentinvention are hazardous but are environmentally friendly.

From the foregoing it should be understood that an object of thecompositions and methods of the present invention is the ability tocarry out any number of specifically tailored oxidation and/or reductionreactions. Another object of the present invention is the ability tocarry out such oxidation and/or reduction reactions far more cheaply andeasily because they require relatively inexpensive and easily obtainableraw materials, such as white rot fungi.

Yet another object of the present invention is the ability to carry outsuch oxidation/reduction reactions using fungi and conditions whichaffect the production of mediators (such as veratryl alcohol) andreductants (such as oxalate).

More specifically, an object of the present invention is the ability todesign a reaction sequence by altering the identities and amounts of theconstituent materials to alternatively reduce and/or oxidize certaintargeted substrates depending on the desired result. Moreover, suchoxidation and reduction reactions can be carried out without having togreatly alter the reaction conditions, such as continuously adding moreoxidant or reductant because such agents are continuously regenerated.

Yet another object of the compounds and methods of the present inventionis the targeted degradation of a variety of recalcitrant environmentalpollutants such as PCBs, chlorinated hydrocarbons, and other toxicorganic wastes.

Still, a further object of the compositions and methods of the presentinvention is the ability to generate oxygen necessary to sustain thelife of organisms which must often be employed in highly anaerobicconditions, such as in the case of remediation sites where the organismsare injected deep into contaminated soil.

Additional objects and advantages of the invention will be set forth inthe description which follows, and in part will be obvious from thedescription, or may be learned by the practice of the invention. Theobjects and advantages of the invention may be realized and obtained bymeans of the instruments and combinations particularly pointed out inthe appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of the total general reaction scheme of thepresent invention (with the understanding that not all of the reactionsshown therein must necessarily be employed in all situations).

FIG. 2. ESR spectra of PBN-CCl₃ radical adducts. (A) The reactionmixture contained lignin peroxidase H2. H_(2u) O₂ veratryl alcohol,EDTA, CCl₄ and PBN (see Material and Methods for concentrations). (B)The conditions were the same as described in (A) except that ¹³ CCl₄ wassubstituted for CCl₄. The spectrometer settings were: scan range 100 G,modulation amplitude 10 G, gain 1×10⁴, microwave power 50 mW,, timeconstant 0.128 sec, scan time 2 minutes.

FIG. 3. ESR spectra of PBN adducts with EDTA derived radicals. (A)Reaction conditions were similar to those described in FIG. 1 exceptCCl₄ was omitted. The spectrometer settings were: scan, range 100 G,modulation amplitude 1.0 G gain 2.5×10⁴, microwave power 50 mW, timeconstant 0.5 sec, scan time 4 minutes. (B) Computer simulated spectra ofthe two major PBN adducts in A. The hyperfine splitting constants usedwere (i) a_(N) =15.6 G, ^(a) _(H) β=3.04 G, (ii) a_(N) =16.3 G, ^(a)_(H) β=4.07 G.

FIG. 4. ESR spectrum of DMPO-superoxide radical adduct. The reactionconditions were similar to those described in FIG. 1 except CCl₄ wasomitted, the pH was 6.5, and DMPO was used as the spin trap. Thespectrophotometer settings were: scan range 100 G; modulation amplitude10 G; gain 8×10³, microwave power 50 mW; time constant 0.128 sec; andscan time 2 minutes; modulation amplitude i 0 G; gain 8×10³, microwavepower 50 mW; time constant 0.128 sec; and scan time 2 minutes.

FIG. 5. The proposed scheme for the reduction of CCl₄ by ligninperoxidase H2. The organic acid is EDTA.

FIG. 6. and FIG. 7 Oxygen production in the presence and absence ofveratryl alcohol. All reaction mixtures contained 100 mM sodium acetate,pH 5.0, 1 mM H₂ O₂ and 0.63 μM LiPH2. The production of O₂ was measuredin the absence of veratryl alcohol (a) with 50 μM veratryl alcohol (VA)added where indicated by the arrows (b and c), when 50 μM veratrylalcohol was added before initiating the reaction with LiPH2 (d) (of FIG.7), and when 3 mM veratryl alcohol was added before initiating thereaction with LiPH2 (e) (of FIG. 7).

FIG. 8 and FIG. 9. Effect of hydrogen peroxide on the rates of LiPH2catalyzed veratryl aldehyde formation and oxygen production. Thereaction mixtures contained 100mM sodium acetate, pH 5.0, 0.63 μM LiPH2,3 mM veratryl alcohol, and the indicated amounts of H₂ O₂. FIG. 8, theeffect of H₂ O₂ concentrations between 0.125 and 40 mM; FIG. 9, theeffect of H₂ O₂ concentrations between 0.125 and 1.5 mM on veratrylaldehyde formation ( ) and O₂ production ( ) . The values in FIG. 9represent the mean of triplicate measurements with error bars indicatingthe standard deviations.

FIG. 10. Effect of oxalate on the rate of LiPH2-catalyzed oxygenconsumption. The reaction mixtures contained 100 mM sodium acetate, pH5.0, 0.63 μM LiPH2, 1 mM H₂ O₂, 3 mM veratryl alcohol, and the indicatedoxalate concentrations. The values represent the mean of triplicatemeasurements. The error bars indicate standard deviations.

FIG. 11. Effect of hydrogen peroxide on the rate of LiPH2-catalyzedoxygen consumption. The reaction mixtures contained 100 mM sodiumacetate, pH 5.0, 0.63 μM LiPH2, 3 mM veratryl alcohol, 10 mM oxalate, pH5.0, and the indicated concentrations of H₂ O₂. All values represent themean of triplicate measurements. The standard deviations are indicatedby the error bars.

FIG. 12. Effect of oxalate on the rate of LiPH2-catalyzed oxygenproduction. The reaction mixtures contained 100 mM sodium acetate, pH5.0, 0.63 μM LiPH2, 1 mM H₂ O₂, 3 mM veratryl alcohol, and the indicatedconcentrations of oxalate. The values represent the mean of triplicatemeasurements. The error bars indicate standard deviation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention relates to compounds and methods for carrying outoxidation reactions, reduction reactions, and mixtures of both dependingon the reaction conditions and constituent chemicals that are added tothe reaction mixture. More specifically, the invention relates tocompounds and methods wherein a free radical generating catalyst is usedto react with a mediator to form the free radical of the mediator. Oftenthis free radical is a cation radical which, being electron deficient,is a strong oxidizing agent. It can be used directly to oxidize, andthereby degrade, a targeted organic substrate, or it can be reacted witha suitable organic chemical (or "reductant") to form the free radical ofthe reductant, commonly an anion radical, which is a strong reducingagent. Using either the cation radical oxidant, the anion radicalreductant, or both, a wide variety of oxidation or reduction reactionsor both may be carried out, such as for the degradation of recalcitranttoxic organic chemicals found, for example, in toxic waste sites.

Under the Superfund laws passed in 1980 ("CERCLA") many remediationsites in the United States must be cleaned up. The pollutants in thesesites include a variety of toxic chemicals, including PCB's, chlorinatedhydrocarbons, and other pernicious substances not generally subject tonormal biodegradation reactions. These substances and others have provenextremely difficult to remove or destroy.

One method is simply to dig up large amounts of earth and put them intotall columns which are heated by air, thereby driving off the chemicals.One obvious shortfall of this method is that the chemicals are merelytransferred from the soil into the air. Under the present invention,substances such as chlorinated hydrocarbons are first reduced to stripoff the chlorine moieties, and then oxidized to CO₂.

In addition, the present invention provides methods for producingmolecular oxygen, which is often necessary to sustain the viability ofliving organisms such as white rot fungi.

I. General Reaction Scheme.

Referring to FIG. 1, the general reaction scheme of the presentinvention is as follows: (1) a peroxidase is used to (2) react with amediator (oxidant precursor) (3) to form the free radical of themediator, which is a strong oxidant; (4) the free radical of themediator then reacts with and oxidizes a reductant (5) to form theradical of the reductant (regenerating the mediator); (6) the freeradical of the reductant is a strong reducing agent capable of reactingwith an electron acceptor (7) which is reduced in the final step of thereaction sequence.

In any given reaction mixture, there may be multiple oxidations andreductions occurring simultaneously depending on the constituentmaterials, their concentrations, and the pH of the system. It should beunderstood that the reductant described in step 4 has heretofore beenviewed as merely an inhibitor of the oxidative nature of the freeradical of the mediator. Not until this invention has it been understoodthat the reductant can actually be converted into a powerful reducingagent. Shah., M. et al., On the Mechanism of Inhibition of the VeratrylAlcohol Oxidase Activity of Lignin Peroxidase H2 by EDTA, J. Biol.Chem., 267:21564-69 (October 1992). For purposes of disclosure thisarticle is incorporated herein by specific reference. It is thisunderstanding of the nature of the reductant that allows one to design areaction sequence which can specifically target selected organicsubstances for degradation.

For example, in the case of chlorinated hydrocarbons, the molecules mustfirst be reduced to remove the chlorine moieties (which are reduced tochloride ions). In the experiments set forth below, CCl₄ was chosenbecause of its extreme stability and resistance to degradation. Once itwas learned that a reaction sequence could specifically be designedwhich would reduce CCl₄ to trichloromethyl radical (which is thendegraded to CO₂).

Once the chlorine moieties have been removed from the chlorinatedhydrocarbon, the remaining carbon containing molecules are easilyoxidized to CO₂ by means of the free radical of the mediator.

This method of dehalogenation is contrary to the current understandingthat LiP alone actually catalyzes the halogenation of organic molecules.Nevertheless, halogenation reactions are inhibited and actually reverseto create dehalogenation conditions according to the present invention.Shah, M. et al, Oxidation of Halides by Peroxidases and Their SubsequentReactions, Arch. Biochem. Biophys., 300:000 (January 1993). For purposesof disclosure this article is incorporated herein by specific reference.

In addition to the free radical of the mediator, hydroxyl radicals, themost powerful oxidant known, can be produced according to the presentreaction. A more thorough discussion of how to generate hydroxylradicals is set forth in Barr, D. et al, Production of Hydroxy Radicalby Lignin Peroxirane, Arch. Biochem. Biophys., 298:480-85 (November1992). For purposes of disclosure, this article is incorporated hereinby specific reference. This article demonstrates that the hydroxylradical is obtained after the final step of the general reactionsequence wherein molecular oxygen is reduced to superoxide anion radicalduring the decarboxylation of the anion radical of either EDTA oroxalate. Superoxide is known to catalyze the production of hydroxylradical in the presence of ferric ion via an iron dependent Haber-Weissreaction. This article confirms that this reaction also occurs under thereaction conditions of the present invention.

Finally, the presence of molecular oxygen where none was initiallypresent led to another study to be published in a paper by Barr, D. etal, entitled Veratryl Alcohol-dependent Production of Molecular Oxygenby Lignin Peroxidase, J. Biol. Chem., 268:14 (Jan. 1993). The studiesset forth in this unpublished article demonstrate that both superoxideand molecular oxygen can be produced where hydrogen peroxide is thereductant which reacts with veratryl alcohol. By this mechanism hydrogenperoxide is oxidized to superoxide molecule, which then dismutates intomolecular oxygen and hydrogen peroxide. However, whether molecularoxygen is consumed or produced is dependent upon the concentration ofhydrogen peroxide (and other reductants such as oxalate), which is alsonecessary in many cases to activate the peroxidase to begin the reactionsequence. More specific details regarding these findings are set forthbelow.

II. Preferred Constituents and Their Reactions.

Preferred peroxidases include LiP, horse radish peroxidase, andlactoperoxidase. Each of these peroxidases are activated by hydrogenperoxide to form an activated 2-electron oxidized enzyme intermediate.It is this enzyme intermediate, being electron deficient, which reactswith the mediator to form the free radical of the mediator. In fact,because the enzyme intermediate is deficient of two electrons, one moleof the enzyme intermediate is normally able to react with two moles ofmediator to form two moles of the free radical of the mediator.

Preferred mediators include veratryl alcohol, ABTS, Mn II, iodide,1,2,3-trimethoxybenzene, 1,4-dimethoxybenzene, and1,2,4-trimethoxybenzene. Each of these mediators is oxidized by theenzyme intermediate to form a free radical of the mediator. In the caseof veratryl alcohol, methoxybenzenes, or ABTS, the cation radical isformed. The use of iodide or Mn II provides a method for the formationof neutral radicals, while the use of any of the others provides amethod for the formation of cation radicals of different redoxpotentials. In addition, in the case of ABTS the cation free radical canbe formed by non-enzymatic, electrochemical means. However, the use ofenzymes allows for more carefully controlled and economical productionof radicals (either oxidative or reductive).

Preferred reductants include EDTA, oxalate, hydroquinones, hydrogenperoxide, and quinones plus quinone reductases. Of the reductants, onlyoxalate, quinone with quinone reductase, and hydrogen peroxide aresecreted by white rot fungi. Depending on the desired reactions, thisfact must be taken into consideration, either to take advantage orinhibit the reactions which involve either oxalate or hydrogen peroxide.(The same is true for veratryl alcohol, which is also secreted by whiterot fungi.)

One mole of reductant is oxidized by one mole of the free radical of themediator to form one mole of the free radical of the reductant. In eachcase the anion radical of the reductant is formed. The anion radicals ofeither EDTA or oxalate are further oxidized during the next reactivestep with the electron acceptor and decarboxylated. The anion radical ofhydrogen peroxide (or superoxide) is further oxidized to molecularoxygen. Finally, the anion radical of the hydroquinones (semiquinones)are further oxidized to their quinone analogs during the final reactivestep of the reaction sequence of the present invention.

In the case of the use of manganese dependent peroxidases, wherein Mn IIis the mediator and is oxidized to Mn III, preferred reductants will beeither hydroquinones or quinones plus quinone reductase to catalyze thereductive reactions.

Suitable electron acceptors, which are reduced by the anion radicals ofthe reductant, can be any organic molecule which is targeted fordegradation but is usually a halogenated hydrocarbon such as CCl₄, DDT,TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) (commonly known as "dioxin"),lindane (1,2,3,4,5,6,-hexachlorocyclohexane), and PCBs. In addition,molecular oxygen can act as the electron acceptor and be converted tosuperoxide, a powerful reductant, which in turn can reduce chlorinatedhydrocarbon or further react with ferric ion via Haber-Weiss reactionsto form the hydroxyl radical, a powerful oxidant. Other electronacceptors which have been proven to work include NBT and cytochrome c.

In the case where hydrogen peroxide was the reductant, and superoxidedismutates (or reacts with itself) to form hydrogen peroxide andmolecular oxygen, one equivalent of superoxide is acting as the reducingagent, while a second equivalent of superoxide is acting as the electronacceptor. The first equivalent of superoxide is oxidized to molecularoxygen, while the second equivalent of superoxide is reduced to hydrogenperoxide. In this manner, the inclusion of hydrogen peroxide as thereductant still follows the generalized reaction scheme of the presentinvention.

From the foregoing, it should be understood that the reactions of thepresent invention allow for the formation of superoxide, hydroxyl,iodide, organic acid, semiquinone radicals, and combinations of any ofthem, of very different redox potentials. Hence, the present inventionis suitable for catalyzing both oxidative and reductive reactions on avariety of organic substrates.

III. Reactions Involving White Rot Fungi.

White rot fungi, such as Phanerochaete chrysosporium, are known fortheir ability to degrade lignin to carbon dioxide. Some of the importantcomponents of the lignin degrading enzyme system of the fungi are ligninperoxidase, veratryl alcohol and H₂ O₂. In addition to lignin, the fungiare known to mineralize a variety of recalcitrant chemicals such asCCl₄, DDT, TCDD, Lindane, and PCBs to carbon dioxide. Although white rotfungi were previously shown to be able to degrade halogenated chemicals,the mechanism was heretofore never understood. Hence, controlled,sustainable reactions could not be carried out on a large scale, such asunder conditions relating to the remediation of toxic waste sites. Inparticular, dehalogenation by LiP alone has never been demonstrated. Infact, LiP has been reported to be a haloperoxidase. Ranganathan, V. etal., Biochemistry, 26:5127-32 (1987); Farhangrazi Z. et al.,Biochemistry, 31:10763-68 (1992).

Therefore, the focus of most researchers has been towards the oxidativereactions of LiP instead of the reductive reactions of LiP of thepresent invention. Because chlorinated organic materials are highlyelectron deficient they cannot be oxidized by the enzyme or the freeradical of the mediator. For the reductive dehalogenation of thesechemicals, reducing reaction conditions are required. According to thepresent invention it has been discovered that LiP is able to catalyzereduction reactions using organic acids like EDTA or oxalate as areductant and veratryl alcohol as a free radical mediator. Shah, M. etal., On the Mechanism of Inhibition of the Veratryl Alcohol OxidaseActivity of Lignin Peroxidase H2 by EDTA, J. Biol. Chem., 267:21564-69(October 1992); Barr, D. et al., Production of Hydroxyl Radical byLignin Peroxidase From Phanerochaete chrysosporium, Arch. Biochem.Biophys., 298:480-85 (November 1992).

These studies demonstrated the oxidation of organic acids to anionradicals by veratryl alcohol cation radical, produced by LiP. Theorganic acid radicals were shown to reduce various electron acceptorssuch as cytochrome c, NBT, ferric iron and molecular oxygen. Thisfinding led to the investigation into the possible reduction ofhalogenated organic materials by a similar mechanism. In this study CCl₄was used as a representative halogenated organic material as it is awell known electron acceptor.

The data presented herein indicate that CCl₄ is first reduced totrichloromethyl radical by the reductive reactions of lignin peroxidasein a reaction mixture containing lignin peroxidase H2, veratryl alcohol,H₂ O₂, and EDTA. These results suggest that the reduction of CCl₄ totrichloromethyl radical is a free radical mediated, three-step process,where veratryl alcohol is the free radical mediator, EDTA the reductant,and CCl₄ an electron acceptor.

The first step is the oxidation of veratryl alcohol to the veratrylalcohol cation radical by LiPH2; the second step is the oxidation ofEDTA to the anion radical of EDTA by the veratryl alcohol cationradical; and the third step is the reduction of CCl₄ to thetrichloromethyl radical by the EDTA anion radical, with the concomitantoxidation and decarboxylation of EDTA. The trichloromethyl radical,which is far more unstable than CCl₄, is subsequently dehalogenatedunder the reductive conditions which are present. Thus, it is nowunderstood for the first time that the reduction of halogenated organicmaterials can be carried out using LiP as long as a sufficient amount ofreductant is present to react with the free radical of the mediator.Based on this novel finding, as well as other knowledge gathered by thestudies relating to the production of molecular oxygen, the generalizedscheme set forth above is proposed for the degradation of halogenatedorganic chemicals by P. chrysosporium.

EXPERIMENTAL PROCEDURES

Materials: Hydrogen peroxide, PBN and Tempol were obtained from SigmaChemical Company (St. Louis, Mo.). Veratryl alcohol and DMPO werepurchased from Aldrich Chemical Company (Milwaukee, Wis.). EDTA waspurchased from Mallinckrodt (Paris, Ky.). Buffers and reagents wereprepared with purified water (Barnstead Nanopure 11 system specificresistance 18.0 megohm - cm). Carbon tetrachloride was purchased from EMScience (Gibbstown, N.J.). Carbon-13 labeled CCl₄ was purchased from MSDIsotopes (Montreal, Canada).

Lignin Peroxidase H2 Production and Purification: Culture conditions forthe production of lignin peroxidase H2 from P. chrysosporium and itspurification were as previously described in Tuisel, H. et al, Arch.Biochem. Biophys, 279:158-66 (1990). For purposes of disclosure, thisarticle is incorporated by specific reference. Lignin peroxidase H2 (75U/mg) was used for all the experiments.

Spin Trapping of Trichloromethyl, EDTA and Superoxide Radicals: Thetrichloromethyl radical was detected by electron spin resonance (ESR)spectroscopy as its spin adduct with PBN. Reaction mixture contained0.1M chelex treated sodium phosphate buffer, pH 6.0, 25 μM ligninperoxidase H2, 4 mM EDTA, 500 μM H₂ O₂, 1% CCl₄, 90 mM PBN, and 1 mMveratryl alcohol. Spectral recording began within 1 minute following theinitiation of reaction with 500 μM H₂ O₂. ESR spectra were recorded atroom temperature using a Varian E-109 spectrometer operating at 9.5 GHzwith 100 KHz modulation frequency. Hyperfine splitting constants weredetermined by comparison with the standard Tempol using 17.1 G for a_(N)in water.

The EDTA derived radicals were detected under similar conditions inreaction mixtures without CCl₄. Superoxide radicals were detected inthese reaction mixtures without CCl₄ by increasing the pH to 6.5 andusing 60 mM DMPO as the spin trap.

Anaerobic reactions were performed by purging each component of thereaction mixture, the reaction mixture itself, and the ESR cavity withnitrogen gas for 10 minutes.

As indicated by FIG. 2A, LiPH2 reduced CCl₄ to the trichloromethylradical in a reaction mixture containing H₂ O₂, veratryl alcohol, EDTA,and PBN. The ESR splitting constants of the spin adduct were a_(N) =14.0G, a_(H).sup.β =1.8 G. These values are in agreement with those reportedpreviously for the PBN-trichloromethyl radical spin adduct. Janzen, E.et al., Environ.. Hlth, Persp., 64:151-70 (1985) ("Janzen et al"). Tofurther confirm the identity of the trichloromethyl radical, ¹³ CCl₄ wasused. The ESR spectrum of PBN spin adduct is shown w in FIG. 2B. The ESRsplitting constants were found to be a_(N) =14.0 G, a.sub.β^(H) =15 G,and a.sub.β¹³ C=9.6 G. These values are also in agreement with thosereported previously for the PBN-¹³ C-labeled trichloromethyl radicalspin adduct. Janzen et al.

Upon removal of CCl₄, the ESR spectrum was a mixture of carbon-centeredradicals (FIG. 3A). Computer simulation of the two major spin adductsfrom these spectra is shown in FIG. 2B. The hyperfine splittingconstants used in this simulation were a_(N) =15.6 G, a_(H).sup.β =3.04G for one radical, and a_(N) =16.3 G, a_(H).sup.β =4.07 G for the other.These values were the same as that reported by us for EDTA radicals.Shah et al., J. Biol. Chem., 267:21564-69 (1992). When DMPO was used asa spin trap, superoxide radicals were trapped in a reaction mixturecontaining lignin peroxidase H2, H₂ O₂, EDTA and veratryl alcohol (FIG.4). The hyperfine splitting constants were a_(N) =14.3 G, a_(H).sup.β=11.7 G, a_(H) 1.3 G. These values are in agreement with values in theliterature. Buettner, G., Free Rad. Biol Med., 3:259-303 (1987). NoDMPO-superoxide adduct was formed upon removal of enzyme, H₂ O₂,veratryl alcohol or EDTA.

To check whether the EDTA radicals or superoxide was involved in thereduction of CCl₄, the reaction was studied under anaerobic conditions.PBN-trichloromethyl radical adducts were observed indicating EDTAradicals were able to reduce CCl₄.

The proposed scheme for the reduction of CCl₄ by lignin peroxidase H2 inthe reaction mixture containing EDTA, veratryl alcohol and H₂ O₂ shownin FIG. 5 is as follows. First, veratryl alcohol is oxidized to theveratryl alcohol cation radical by lignin peroxidase H2 in the presenceof H₂ O₂. Then, the organic acid (i.e., EDTA) is oxidized to an organicacid radical by the cation radical of veratryl alcohol. The organic acidradical reduces CCl₄ to the trichloromethyl radical which results in thedecarboxylation of the organic acid.

The proposed scheme is in agreement with previously reported mechanismsfor the reductive reactions of lignin peroxidases. Shah, M. et al., J.Biol. Chem., 267:21564-69 (1992); Barr, D. et al., Arch. Biochem.Biophys., 298:480-85 (1992). It was shown that the cation radical ofveratryl alcohol serves as the oxidant in the oxidation of EDTA to EDTAderived radicals, and the EDTA derived radicals in turn, serve as thereductant for the reduction of various electron acceptors, such ascytochrome c, NBT, molecular oxygen and ferric iron. In the presentstudy, it was found that CCl₄ serves as an electron acceptor in thisreductive system and was reduced under anaerobic, as well as aerobicconditions. EDTA radicals are proposed to be the reductant in thereduction of CCl₄ to the trichloromethyl radical.

The proposed scheme for the degradation of CCl₄ is also in agreementwith the oxidative reactions of lignin peroxidase previously known inthe art. Based on teachings known in the art it would seem safe toconclude that the oxidation of aminotriazole, α-keto- -methiol-butyricacid, and malonic acid reductants by LiP should occur in the presence ofthe veratryl alcohol cation radical. That veratryl alcohol istransformed into the cation radical is confirmed by Gilardi, G. et al.,Biochem. Biophys Acta., 1041:129-32 (1990). For purposes of disclosurethis article is incorporated herein by specific reference.

The proposed scheme has two major advantages with respect to thedegradation of halogenated organic materials by white rot fungi. First,it provides an approach to inhibit the proven haloperoxidase reactionsof LiP. It was also shown that the iodinating species (HOI or EOI, whereE is enzyme) produced by peroxidases reacts with EDTA. In the process,EDTA was decarboxylated. Secondly, it suggests an approach for thedegradation of highly halogenated organic materials. In this study weshow that organic acid radicals could serve as the reductant for thereduction of CCl₄. In addition to superoxide and organic acid radicals,hydroxyl radicals can also be formed upon addition of ferric iron viathe proposed oxidoreductive reactions of LiP. The degradation ofhalogenated organic materials by hydroxyl radicals is anotheralternative.

Among the components involved in the proposed scheme, EDTA is the onlyone which is not physiologically relevant. Future research is requiredto identify fungal reductants similar to EDTA. Oxalic acid and otherorganic acids are produced by white rot fungi. It should also be notedoxalic acid itself does not have the site for its halogenation.Haloalkanes are reported to react with amines upon photochemicalactivation. So, other potential physiologically relevant reductantswould include amino acids which are functionally similar to EDTA. It isknown that white rot fungi excrete proteases and the degradation ofprotein by proteases might be a possible source of such amino acids.

Another important finding was that the reduction of CCl₄ was performedin an aqueous media although organic radicals are considered to behydrophobic. Hence, the reduction of CCl₄ would be expected to occuronly in the organic phase, i,e., the CCl₄ phase. It is suggested thatradicals are somehow being transferred from the aqueous phase to theCCl₄ phase. Since lignin is also hydrophobic like CCl₄, organic radicalsproduced by LiP probably attack lignin via a similar free radicalmediated process.

It is also significant that like CCl₄, lignin is also a highly oxidizedmolecule. Therefore, the first step in the degradation of lignin mightinvolve a reduction reaction by reducing radicals produced by LiP.Therefore, although not previously understood, it is highly probablethat the degradation of lignin involves a combination of reductive andoxidative steps according the reactive scheme proposed by the presentinvention.

IV. Studies Relating to the Generation of Molecular Oxygen.

Although reported in the literature that veratryl alcohol prevents thedeactivation of LiP in the presence of excess hydrogen peroxide, nothingwas known regarding this mechanism. See Khadar, V. et al., Biochem.,29:8535-39 (1990). In fact, studies leading up to the present inventionshowed that hydrogen peroxide can react with the cation radical ofveratryl alcohol to form molecular oxygen via the formation ofsuperoxide. This shows that hydrogen peroxide does not interfere withveratryl alcohol mediated LiP oxidations by reacting with LiP; ratherexcess hydrogen peroxide interferes with the oxidation reactions byreaction with the cation radical of veratryl alcohol, a powerful oxidantwithin the reactive scheme of the present invention. The result is theformation of superoxide anion radical, itself a powerful reductantrather than an oxidizer, while veratryl alcohol is reduced to its nativestate. Moreover, at least part of the hydrogen peroxide is regeneratedupon the dismutation of superoxide to,hydrogen peroxide and molecularoxygen. The proposed reaction scheme for the reaction of hydrogenperoxide and the cation radical of veratryl alcohol is as follows:

    VA.sup.+ ·+H.sub.2 O.sub.2 →VA+O.sub.2 --+2H.sup.+(Eq. 1)

    O.sub.2 --+HO.sub.2 ·+H.sup.+ →O.sub.2 +H.sub.2 O.sub.2 (Eq. 2)

EXPERIMENTAL PROCEDURES

Chemicals--Hydrogen peroxide, oxalate, and superoxide dismutase werepurchased from Sigma. Veratryl alcohol (3,4-dimethyoxybenzyl alcohol),anisole (methoxybenzene), 1,2,3-trimethoxybenzene, 1,4-dimethoxybenzene,and 1,2,4-trimethoxybenzene were purchased from Aldrich.1,2,4,5-Tetramethoxybenzene was synthesized using the procedure ofKersten et al., J. Biol. Chem, 260:2609-12 (1985). The sodium acetatebuffer was prepared using purified water (Barnstead NANO pure II system;specific resistance 18.0 Mohm-cm⁻¹).

Enzyme Production and Purification--The culture conditions used toproduce lignin peroxidases and their purification and activity assaywere as described previously in Tuisel, H. et al., Arch. Biochem.Biophys., 279: 158-66 (1990) ("Tuisel et al."). The extracellular fluidwas dialyzed overnight against 10 mM sodium acetate buffer, pH 6.0, andthe proteins purified on a Mono Q HR 5/5 column (Pharmacia LKBBiotechnology Inc.). Veratryl alcohol oxidase activity in theextracellular fluid was determined by measuring an increase inabsorbance at 310 nm (veratryl aldehyde formation) as describedpreviously in Tuisel et al. The major lignin peroxidase (LiPH2) was usedthroughout this study.

Oxygen Consumption or Production Experiments--Oxygen consumption orproduction was measured using a Gilson oxygraph monitor equipped with a1.8-ml water jacketed reaction chamber and a Clark type oxygen sensitiveelectrode. All reaction mixtures contained 0.63 μM LiPH2, 100 mM sodiumacetate, pH 5.0, 3mM veratryl alcohol, and H₂ O₂ and oxalateconcentrations as indicated in the figure legends. The reactions wereinitiated by the addition of LiPH2. All reactions were performed at roomtemperature. In the experiment involving superoxide dismutase, 98units/ml superoxide dismutase was added approximately 1 min after thereaction was initiated with LiPH2. The reactions involving superoxidedismutase were performed at pH 6.8 because its catalytic effect on thedismutation of O₂ is more marked at pH 6.8 than pH 5.0.

Spectral Characterization of LiPH2--The oxidation state of LiPH2 wasdetermined by scanning between 600 and 480 nm. The concentrations of thereactants are described in the figure legends. All spectral recordingswere made using a Shimadzu UV-160 spectrophotometer.

The effect of veratryl alcohol on LiPH2-catalyzed O₂ production is shownin FIGS. 6 and 7. Oxygen production was observed in the absence ofveratryl alcohol (a). However, the addition of 50 μM veratryl alcohol atvarious times after initiating the reaction enhanced both the rate andextent of O₂ production (b, c, and d of FIG. 7) but to various degrees.When a saturating amount of veratryl alcohol (i,e., 3 mM) was added atthe start of the reaction the O₂ production rate was 6 μM/min (e) (ofFIG. 7).

The effects of H₂ O₂ on veratryl alcohol oxidation and the evolution ofmolecular oxygen are presented in FIGS. 8 and 9. The rate of O₂evolution and the inhibition of veratryl alcohol oxidation appear to beinversely related with respect to the H₂ O₂ concentration. FIG. 8 showsthe effect of H₂ O₂ concentrations between 0.125 and 40 mM on the ratesof veratryl aldehyde formation and O₂ production. A more detailed studywas performed using H₂ O₂ concentrations between 0.125 and 1.5 mM and ispresented in FIG. 9. It has been reported that high concentrations of H₂O₂ result in the inactivation of LiP. Wariishi, H. et al., J. BiolChem., 266:20694-99 (1991). To ensure that the inhibition of veratrylaldehyde formation (at H₂ O₂ concentrations between 0.125 and 1.5 mM)was not due to the formation of an inactive form of LiPH2, the visibleabsorbance spectra of LiPH2 were examined in reaction mixturescontaining 14.7 μM LiPH2 and the same ratios of H₂ O₂ /LiPH2 andveratryl alcohol/LiPH2 as were used in the previous experiment. Theabsorption spectra using a saturating concentration of veratryl alcoholindicated that LiPH2 was always in its activated state. For example,using 23 mM H₂ O₂ and 72 mM veratryl alcohol (equivalent to 0.63 μMLiPH2, 1 mM H₂ O₂ and 3 mM veratryl alcohol, FIGS. 6 and 7 theabsorption maxima were at 528 and 554 nm. However, using lowerconcentrations of veratryl alcohol (23 mM H₂ O₂ and only 575 μM veratrylalcohol) which was equivalent to 0.63 μM LiPH2, 1 mM H₂ O₂, and 50 μMveratryl alcohol (FIGS. 6 and 7) the inactive form of LiPH2 wasdetected. The ability of other methoxybenzenes to mediateLiPH2-catalyzed O₂ production is shown in Table I.

                  TABLE I                                                         ______________________________________                                        Production of Molecular Oxygen by LiPH2 in the                                Presence of H.sub.2 O.sub.2 using Various Electron Mediators                                                Rate                                            Electron mediator E.sub.1/2   μM/min                                       ______________________________________                                        Veratryl alcohol  NA          6.7 ± 0.7                                    Anisole (methoxybenzene)                                                                        1.76        ND                                              1,2,3-Trimethoxybenzene                                                                         1.42        4.8 ± 0.5                                    1,2,4-Trimethoxybenzene                                                                         1.12        4.1 ± 0.5                                    1,2,4,5-Tetramethoxybenzene                                                                     0.81        ND                                              ______________________________________                                         The reaction mixtures contained 100 mM sodium acetate, pH 5.5, 0.8 μM      LiPH2, 1 mM H.sub.2 O.sub.2 and 3 mM electron mediator. The values for        E.sub.1/2  were obtained from reference 25.                                   (NA, not available; ND, not detectable.)                                 

It was postulated that the evolution of oxygen was due to the oneelectron oxidation of H₂ O₂ by the cation radical of veratryl alcohol toform superoxide (O₂ ⁻ ·) and its subsequent dismutation to yield O₂.Thus, it would be expected that superoxide dismutase would enhance therate of O₂ evolution via this mechanism. When 98 units/ml superoxidedismutase was added to reaction mixtures containing LiPH2, H₂ O₂, andveratryl alcohol, the rate of O₂ evolution was increased to 11.8 μM/minas compared with 4.1 μM/min in the absence of superoxide dismutase. Theaddition of boiled superoxide dismutase had no effect on the rate of O₂evolution.

It has been previously reported that oxalate, which is produced by P.chrysosporium, can serve as an electron donor for the reduction of theveratryl alcohol cation radical. E.g., Akamatsu, Y. et al., FEBS Lett.,269:261-63 (1990).

The oxidation of oxalate by the veratryl alcohol cation radical producesthe carbon dioxide anion radical (CO₂ --). The carbon dioxide anionradical reduces molecular oxygen to O₂ --. Thus, oxygen consumption wasobserved in reaction mixtures containing LiP, H₂ O₂, veratryl alcohol,and oxalate. The effect of the oxalate concentration on O₂ consumptionin reaction mixtures containing LiPH2, H₂ O₂, and veratryl alcohol isshown in FIG. 10. It should be noted that some O₂ production is observedwhen no oxalate was added to the reaction mixture. Therefore, the zeropoint on the abscissa was left out of FIG. 10.

Since it appeared that H₂ O₂ also reduced the veratryl alcohol cationradical to produce O₂ -- and subsequently O₂, it would be expected thathigher H₂ O₂ concentrations would inhibit O₂ consumption in reactionmixtures containing oxalate. FIG. 11 shows the effect of increasing theH₂ O₂ concentration on the rate of O₂ consumption in reaction mixturescontaining LiPH2, veratryl alcohol, and oxalate. Indeed, the rate of O₂consumption decreased with increasing concentrations of H₂ O₂. Theeffect of increasing oxalate concentration on the rate ofLiPH2-catalyzed O₂ production is presented in FIG. 22. The rate of O₂production was inhibited by increasing concentrations of oxalate.

The results of this investigation demonstrate that LiPH2 can catalyzethe one electron oxidation of H₂ O₂ using veratryl alcohol as anelectron mediator. The O₂ -- produced by this reaction dismutates to O₂and H₂ O₂. Therefore, the net reaction catalyzed by LiPH2 is theproduction of O₂ from H₂ O₂. This mechanism of O₂ production was furtherevidenced by the fact that superoxide dismutase stimulated the rate ofO₂ evolution. The production of oxygen in the absence of veratrylalcohol might be due to reactions involving the inactive form of LiPH2.When catalytic amounts of veratryl alcohol (i.e., 50 μM) were added, thehighest rate of O₂ production was obtained. This indicated that therewere two mechanisms for O₂ production by LiPH2: one involving theinactive form of LiPH2 and one involving the veratryl alcohol cationradical.

Evidence for this arises from the fact that when the rate of O₂production in the absence of veratryl alcohol (6.5 μM/min) was added tothe O₂ production rate with saturating concentrations of veratrylalcohol (i,e., 3 mM veratryl alcohol gave 6.0 μM/min), a rate of 12.5μM/min was obtained. The rate of O₂ production in the presence of acatalytic amount of veratryl alcohol (i.e. 50 μM gave 12.0 μM/min, aboutthe sum of these two rates.

Veratryl aldehyde formation was inhibited by H₂ O₂ apparently by the oneelectron reduction of the veratryl alcohol cation radical back toveratryl alcohol; not by inactivation of the enzyme. The visibleabsorbance spectrum of LiPH2 in this reaction mixture showed that theenzyme was in the compound II state. Therefore, the inhibition ofveratryl aldehyde formation at concentrations of H₂ O₂ between 0.125 mMand 1.5 mM ,was not due to the inactivation of LiP (i.e., formation ofcompound III). It was also found that other methoxybenzenes, which areoften considered lignin model compounds, could serve as the electronmediator for LiPH2-catalyzed O₂ production. The ability of thesemethoxybenzenes to mediate LiP-catalyzed O₂ production was related tothe redox potential of the methoxybenzene. The redox potential for theone electron oxidation of H₂ O₂ to O₂ -- is 0.89 V. The redox potentialof 1,2,4,5-tetramethoxybenzene is 0.81 V. Accordingly, no O₂ productionwas observed when 1,2,4,5-tetramethoxybenzene was used as the electronmediator. Although the redox potential of anisole (methoxybenzene) issufficient to promote the oxidation of H₂ O₂ (1.76 V), it is not asubstrate for lignin peroxidase. Therefore, no O₂ production wasobserved in reaction mixtures containing anisole. The othermethoxybenzenes have the appropriate redox potential for oxidation of H₂O₂ and O₂ evolution was observed when these were used with LiP and H₂O₂.

In an earlier study by Magnusson et al., J. Biol. Chem., 259:197-205(1984), O₂ evolution was observed following the oxidation of iodide tohypoiodite by thyroid peroxidase. It was concluded that O₂ evolution wasdue to the oxidation of H₂ O₂ by hypoiodite. Studies from our laboratoryshow that O₂ is produced following the oxidation of iodide by LiPH2. Thereaction of hypoiodite with H₂ O₂ to produce molecular oxygen isconsidered to be a two-electron process. Shah, M. et al, Oxidation ofHalides by Peroxidases and Their Subsequent Reductions, Arch. Biochem.Biophys., 300:000 (January 1993). However, the veratrylalcohol-dependent O₂ production arises from the one-electron oxidationof H₂ O₂ by the veratryl alcohol cation radical to produce O₂ --. Othermethoxybenzenes, which are substrates for LiP, also served as theelectron mediator for O₂ production. These methoxybenzenes also formcation radicals when they are oxidized by LiP.

Other investigations have shown that electron donors such as oxalate andEDTA can reduce the veratryl alcohol cation radical back to veratrylalcohol. Studies by Akamatsu, Y. et al., FEBS Lett., 269:261-63 (1990)demonstrated that oxalate was a noncompetitive inhibitor of LiPcatalyzed veratryl alcohol oxidation. The CO₂ -- produced by theoxidation of oxalate has been shown to consume molecular oxygen.

    VA.sup.+ ·+oxalate→VA+CO.sub.2 --+CO.sub.2 (Eq. 3)

    CO.sub.2 --+O.sub.2 →CO.sub.2 +O.sub.2 --           (Eq. 4)

In the present study it was found that O₂ consumption by this mechanismcould be inhibited by increasing concentrations of H₂ O₂. The inhibitionappears to be due to the competing reaction of H₂ O₂ with the veratrylalcohol cation radical. It was also found that the rate ofLiPH2-catalyzed O₂ production was inhibited by increasing concentrationsof oxalate. Therefore, it was concluded that if oxalate donated anelectron to the veratryl alcohol cation radical, oxygen consumptionwould result due to reactions 3 and 4. However, if H₂ O₂ was theelectron donor, the result was oxygen production via reactions 1 and 2.These results imply that the rates of O₂ production or O₂ consumption byLiPH2 are dependent on the relative concentrations of H₂ O₂ and oxalate(or possibly other organic acids). The importance of the O₂concentration in lignin degradation by P. chrysosporium has beendemonstrated in previous studies. It was found that flushing cultures ofP. chrysosporium with 100% O₂, as opposed to air, enhanced the rate oflignin degradation. Since P. chrysosporium produces H₂ O₂, veratrylalcohol, and oxalate, the mechanisms described here may be involved inregulating the O₂ concentration for the degradation of lignin in wood byP. chrysosporium.

V. Summary

From the foregoing it should be understood that any number of oxidationand/or reduction reactions can be carried out depending upon thereaction conditions which are chosen. For example, the reactionmechanisms proposed by the present invention allow for the formation ofsuperoxide, iodide, hydroxyl, organic acid, and hydroquinone radicalswith greatly varying redox potentials. Depending upon the targetedsubstrate the raw materials added to the system can be optimized toyield the most efficient and sustainable reaction sequence.

Through the careful manipulation of the reactants and the reactionconditions, oxidations and reductions can be carried out in a far morepredictable manner than heretofore possible. In addition, because manyof the complex chemicals necessary to carry out the reactions can bederived from simple organisms, such as white rot fungi, the reactionsare far more economically feasible than conventional oxidation andreduction reactions.

Finally, because the oxidation/reduction reactions of the presentinvention can easily be carried out on a large scale basis and aresustainable as long as there exist nutrient targeted substrates, theyare especially well suited to remediate any number of toxic substancesnow found throughout the country in toxic waste sites or otherremediation sites.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

What is claimed and desired to be secured by United States Patent is: 1.A method of selectively oxidizing or reducing a targeted substrate, themethod including the steps of;providing a peroxidase to serve as a freeradical generating catalyst; adding a suitable mediator which can beoxidized by said peroxidase to form the free radical of said mediator,whereby said free radical of said mediator is generated by a reactionbetween said peroxidase and said mediator, said free radical of saidmediator being an oxidizing agent; adding hydrogen peroxide which can beoxidized by said free radical of said mediator to formsuperoxide,whereby said superoxide is generated by a reaction betweensaid free radical of said mediator and said hydrogen peroxide; andexposing at least one of said free radical of said mediator or saidsuperoxide so generated to a targeted substrate, said substrate reactingin a oxidation/reduction reaction with at least one of said free radicalof said mediator or said superoxide.
 2. A method as defined in claim 1wherein said targeted substrate is oxygen and said oxygen is reduced tosuperoxide.
 3. A method as defined in claim 1 wherein said superoxidedismutates to form molecular oxygen.
 4. A method as defined in claim 2wherein said superoxide dismutates to form molecular oxygen.
 5. A methodas defined in claim 1 wherein the peroxidase is provided by a white rotfungus.
 6. A method as defined in claim 1 wherein the mediator isprovided by a white rot fungus.
 7. A method as defined in claim 5wherein the mediator is provided by a white rot fungus.
 8. A method asdefined in claim 1 wherein the hydrogen peroxide is provided by a whiterot fungus.
 9. A method as defined in claim 7 wherein the hydrogenperoxide is provided by a white rot fungus.
 10. A method of producingmolecular oxygen in an oxidation/reduction involving a white rot fungi,the method comprising the steps offproviding a peroxidase to serve as afree radical generating catalyst; adding a suitable mediator which canbe oxidized by said peroxidase to form the free radical of saidmediator, whereby said free radical of said mediator is generated by areaction between said peroxidase and said mediator, said free radical ofsaid mediator being an oxidizing agent; adding hydrogen peroxide as areductant which can be oxidized by said free radical of said mediator toform superoxide, whereby said superoxide is generated by a reactionbetween said free radical of said mediator and said hydrogen peroxide;and allowing said superoxide to dismutate into molecular oxygen andhydrogen peroxide.
 11. A method for producing molecular oxygencomprising the steps of:providing a peroxidase; providing a mediator forreaction with said peroxidase; and providing hydrogen peroxide forreaction with the peroxidase in the presence of the mediator in order toproduce molecular oxygen.
 12. A method as defined in claim 11 whereinhydrogen peroxide is added in an amount sufficient to maintain thehydrogen peroxide concentration in the range of about 1 to about 50 mM.13. A method as defined in claim 11 wherein hydrogen peroxide is addedin an amount sufficient to maintain the hydrogen peroxide concentrationin the range of about 5 to about 25 mM.
 14. A method as defined in claim11 wherein hydrogen peroxide is added in an amount sufficient tomaintain the hydrogen peroxide concentration in the range of about 10 toabout 20 mM.